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M J Higgins

Publications and source records attributed to M J Higgins.

At least 91 records · Page 5Linked to original sources
PubMed1990

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis.

Blotting, Southern↗
PubMed1989

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography.

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Cell Nucleolus↗
PubMed1989

Molecular detection of chromosomal translocations that disrupt the putative retinoblastoma susceptibility locus.

A candidate DNA sequence with many of the properties predicted for the retinoblastoma susceptibility (RB1) locus has been cloned (S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature [London] 323:643-645, 1986). The large size of this gene (ca. 200 kilobases [kb]) and its multiple dispersed exons (Wiggs et al., N. Engl. J. Med. 318:151-157, 1988) complicate molecular screening strategies important in prenatal and presymptomatic diagnosis and in carrier detection. Here we used field inversion gel electrophoresis (FIGE) to construct a restriction map of approximately 1,000 kb of DNA surrounding the RB1 locus and to detect the translocation breakpoints in three retinoblastoma patients. DNA probes from either the 5' or 3' end of the gene were used to detect a 250-kb EagI restriction fragment in DNA from unaffected individuals. Both probes identified an additional hybridizing fragment in the DNA from each patient, permitting the breakpoints in all three to be mapped within the cloned RB1 gene. Analysis of the breakpoint in one translocation cell line allowed the RB1 gene to be oriented with its 5' end toward the centromere. The 5' end of the gene also appeared to be associated with a clustering of sites for several infrequently cleaving restriction enzymes, indicating the presence of an HpaII tiny fragment island. The detection and mapping of the translocation breakpoints of all three retinoblastoma patients to within the putative RB1 gene substantiated the authenticity of this candidate sequence and demonstrated the utility of FIGE in detecting chromosomal rearrangements affecting this locus.

Base Sequence↗
PubMed1989

Pathologic changes in the accessory sex glands of rats treated with a sympatholytic hypotensive agent (losulazine).

Groups of 25 male Sprague-Dawley rats were treated by gastric intubation with either vehicle control (modified methylcellulose) or a suspension of losulazine at 4, 8, 16, or 32 mg/kg/day for 1 yr. Ten rats/group were killed after 6 months of treatment. Reversibility of drug-induced changes was evaluated in 8 rats/group treated for 6 months and held without treatment for 5 months. Daily clinical signs and weekly body weight changes were monitored. Seminal vesicle/coagulating glands were weighed in rats treated for 6 months. Gross and microscopic evaluation of the accessory sex glands (ampullary glands, prostate, seminal vesicles and coagulating glands) was conducted in all rats killed at 6 months, after the recovery period, and in rats that survived through the one-yr treatment period. Ptosis, somnolence, and fecal softening were detected in all groups treated with losulazine. There was a nonreversible body weight gain retardation in groups treated with 8 to 32 mg/kg/day of losulazine for 6 months or 1 yr. Absolute and relative weights of the seminal vesicle/coagulating glands of treated rats were not significantly different from those of control rats. The ventral prostate in a few rats in all treated groups had yellow to tan granular foci. Treatment, but not dose-related sperm granulomas or glandular impaction with inspissated secretion (formation of corpora amylacia) in the ampullary glands, enhanced cellular exudation into the acini of the ventral prostate, and impaction of the seminal vesicles with altered granular to globular secretion were found in rats treated with losulazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Cardiovascular complications of chronic catheterization of the jugular vein in the dog.

Cardiovascular changes associated with indwelling catheters were evaluated in 51 adult beagle dogs catheterized for 4 to 9 weeks. Pathologic changes consistent with traumatic injury were in the vena cava and endocardium of the right atrium of 88% of cannulated dogs. Lesions were characterized by surface denudation and diffuse intimal thickening due to myointimal hyperplasia and deposition of extracellular matrix. Affected intima was lined by hyperplastic, poorly differentiated endothelial cells and contained round to oval cells with characteristics of smooth muscle cells. After 9 weeks, thickened intima was vascularized and composed of spindle-shaped cells and fibrillar stroma. Intimal sclerosis and localized proliferative papillary projections in the vena cava cranial to areas of myointimal hyperplasia occurred infrequently. Traumatic lesions, regardless of location or severity, did not extend below the internal elastic membrane. Inflammatory cellular responses, when present, were minimal. The location, distribution, and morphogenesis of catheter-related cardiovascular lesions distinguishes them from those induced by chemical toxicity or pharmacotoxicity.

Animals↗
PubMed1987

Mechanism of anestrus in rats treated with an antihypertensive agent, losulazine hydrochloride.

The mechanism of anestrus in rats treated with losulazine, a peripheral sympatholytic antihypertensive agent, was investigated by determining its effect on hypothalamic catecholamines and serum sex hormones and by evaluating the influence of bromocriptine on the reproductive functions of rats treated with losulazine. Groups of six female Upjohn Sprague-Dawley rats were treated orally with 10 mg/kg/day of losulazine and/or 18.75 mg/kg/day of bromocriptine for 15 or 27 days. Six rats were treated with losulazine plus 6.25 mg/kg/day of bromocriptine for 16 days followed by losulazine alone for 11 days. Rats treated with losulazine only were depleted of hypothalamic catecholamines, were hyperprolactinemic, and had interrupted estrous cycles and attenuated vaginal mucosa. Treatment with bromocriptine, a dopamine receptor agonist, resulted in suppression of serum prolactin and normal estrous cycles. Rats reverted back to hyperprolactinemia and anestrus shortly after bromocriptine withdrawal. These results suggest that hyperprolactinemia mediated through hypothalamic dopamine depletion is the mechanism of anestrus in rats treated with losulazine.

Anestrus↗
PubMed1987

Cardiovascular alterations in dogs treated with hydralazine.

Groups of 5 male beagle dogs were treated orally with hydralazine tablets in gelatin capsules at a dose of 12 or 24 mg/kg twice a day (6 hours apart) for 2 consecutive days. Five male dogs treated with empty gelatin capsules served as untreated controls. Clinical findings and heart rate changes during treatment and terminal body weight, hematology, and blood chemistry changes were evaluated. The heart, liver, kidneys, spleen, and thymus of each animal were examined microscopically. Dogs in the 12 mg/kg group ate less than control group. Dogs treated with 24 mg/kg did not eat and vomited. Heart rates in both of the treated groups increased by 60% to 80% within 2 hours of treatment and remained high during the entire treatment period. Significant hematologic change was confined to a slight increase in platelet number of dogs treated with 24 mg/kg. Serum glucose was increased in the hydralazine treated dogs. Conjugated serum bilirubin was increased and serum potassium, chloride and phosphorus were decreased in the 24 mg/kg group. Blood urea nitrogen and serum chloride were slightly increased in dogs treated with 12 mg/kg. Treatment-related pathologic alterations were confined to the heart. Two dogs from each of the hydralazine groups experienced acute localized hemorrhage into the epicardium and subepicardium of the right atrium. The media of the muscular branches of the coronary arteries, especially the left coronary artery, was hemorrhagic in 3 dogs from the 24 mg/kg group. Medial necrosis, when seen, tended to be proportional to the severity of medial hemorrhages. There was no necrosis in the papillary muscles of the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Isolation and characterization of an alpha-satellite repeated sequence from human chromosome 22.

We constructed a library in lambda L47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag+/Cs2SO4 gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of alpha-R1-DNA and the X specific alpha-satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived alpha-satellite sequences analogous to other chromosome-specific satellite sequences described previously.

Animals↗
PubMed1985

A gene family in Drosophila melanogaster coding for trypsin-like enzymes.

We have isolated a clustered gene family in D. melanogaster that codes for trypsin-like enzymes. The gene family has been localized to 47D-F by in situ hybridization to polytene chromosomes. The four genes in the family are transcribed in alternating orientations, and code for 1000 nt mRNAs. Transcripts are present at all stages of the life cycle. In situ hybridization to mRNA in tissue sections of third instar larvae showed that transcripts were restricted to the mid-gut. One gene was sequenced. The translated amino acid sequence of the proposed active enzyme is 42% homologous to bovine trypsin. Regions of functional importance are more strongly conserved. These include the active site residues asp102, his57, ser195, and the residue asp189 which is reputed to bind the basic residue at the substrate cleavage site. The activation peptide is not homologous to that of most vertebrate trypsins, suggesting a modified activation mechanism. The sequence further strengthens the hypothesis that the chymotrypsin cleavage specificity developed separately in the vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed1985

Release of immunoreactive Met-enkephalin from the spinal cord by intraventricular beta-endorphin but not morphine in anesthetized rats.

Effect of beta-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin1-13 from the spinal cord was studied in anesthetized rats. Intraventricular beta-endorphin, 16 micrograms, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 micrograms, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin1-13 was not released by either beta-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by beta-endorphin had a retention time identical to [3H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for beta-endorphin and morphine.

Animals↗
PubMed1985

Amplified sequences from chromosome 15, including centromeres, nucleolar organizer regions, and centromeric heterochromatin, in homogeneously staining regions in the human melanoma cell line MeWo.

Homogeneously staining regions (HSRs) in the human melanoma cell line, MeWo, are located on an X and a der(15) chromosome. These regions are homogeneously stained with quinacrine fluorescence, but stain differentially with conventional Giemsa, G-banding, C-banding, and distamycin A/4',6-diamidino-2-phenylindole. There are five and six blocks of positively staining material on the X and der(15) HSRs, respectively. Hybridization in situ with a cloned repetitive Kpn I family member has confirmed the amplification of this sequence along the HSRs. With silver staining of the nucleolar organizer regions (NORs) there appear to be three very strong and two weaker pairs of NORs along the HSR of the X chromosome and four strong and one weaker pair on that of the der(15) chromosome. There was little NOR staining on the normal acrocentric chromosomes in these cells, suggesting preferential transcription of the NORs in the HSRs. Centromere-dot staining revealed a distribution of multiple centromeres similar to the NORs along the two HSRs. These data suggest that a unit composed of the short arm and centromere of chromosome #15 has been amplified and that the HSR present on the X chromosome probably arose by a translocation from chromosome #15.

Cell Line↗
PubMed1985

Organization of a repetitive human 1.8 kb KpnI sequence localized in the heterochromatin of chromosome 15.

We have isolated a repetitive 1.8 kb KpnI DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag+-Cs2SO4 gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5' AATGG 3' similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed.

Chromosomes, Human, 13-15↗
PubMed1984

Isolation of two Drosophila melanogaster genes abundantly expressed in the ovary during vitelline membrane synthesis.

Several genomic clones were isolated from a Drosophila library screened with cDNA prepared from abundant adult female mRNA. Cytoplasmic dot hybridizations have shown that the genes in all of these clones are expressed only in posteclosion (stages 8-14) follicles. One set of overlapping clones (lambda 20, lambda 28, and lambda 30) was localized by in situ hybridization to 66D, a previously described locus for chorion genes. Restriction mapping demonstrated that these clones contained chorion genes which had been isolated previously. Another clone, lambda 7, was mapped to chromosomal region 26A. This clone carries genes that hybridized to mRNA species similar or identical in size to the known chorion genes encompassed by lambda 28. Furthermore, one of these genes shows homology to the 66D chorion locus, apparently with the s18-1 gene. R-loop and S1-nuclease mapping indicated that lambda 7 contains two genes of 700-800 base pairs in length. Dot hybridization of cytoplasmic RNA from egg chambers demonstrated that these genes are expressed predominantly during stages 9 + 10, the time of vitelline membrane synthesis. Analysis of DNA extracted from embryos and various female tissues by dot hybridization showed that lambda 7 sequences are not amplified in the mature ovary. These results suggest that the two genes carried by lambda 7 and derived from region 26A may code for protein components of the vitelline membrane. In addition it appears that some evolutionary relatedness exists between one of these genes and a member of the chorion multigene family.

Animals↗
PubMed1984

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line.

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.

DNA Restriction Enzymes↗
PubMed1981

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.

Animals↗
PubMed1980

Hepatic HMGCoA reductase in human cholelithiasis: effects of chenodeoxycholic and ursodeoxycholic acids.

To study further the role of hepatic cholesterol synthesis in patients with gallstones, the activity of the rate-limiting step in cholesterogenesis, hydroxymethyl glutaryl co-enzyme A reductase (HMGCoAR), was measured in operative wedge liver biopsies from ten patients with untreated cholesterol gallstones, six with pigment stones and ten controls. Hepatic HMGCoAR was also measured in six patients with cholesterol-rich gallstones treated for 1-24 months with 14.6-18.6 mg chenodeoxycholic acid (CDCA) kg-1 day-1, in two patients with radiolucent pigment stones treated with 17.3 and 17.7 mg CDCA kg-1 day-1 and in ten other patients with cholesterol-rich stones given 4.5-7.2 mg ursodeoxycholic acid kg-1 day-1 for 1-6 months. HMGCoAR activity was also related to the free and esterified cholesterol content of both hepatic homogenates and the microsomal fractions. Compared with the controls (HMGCoAR activity 14.6 +/- 1.6 (SEM) pmole mg microsomal protein-1 min-1), the mean activity in untreated cholesterol cholelithiasis was 32.2 +/- 2.0 (P < 0.001), but was near normal in patients with pigment stones (16.2 +/- 1.5). In cholesterol gallstone patients, chenodeoxycholic acid treatment reduced the mean enzyme activity by 51% compared with the untreated gallstone group (P < 0.001) and in smaller doses, ursodeoxycholic acid therapy lowered it by 40% (P < 0.001) but in the two patients with pigment stones, CDCA did not seem to affect HMGCoAR activity. Despite this four-fold variation in enzyme activity, there were no significant differences in mean free or esterified cholesterol concentrations in whole liver homogenates or in the microsomal fraction from any of the patient groups.

Adolescent↗